1. Field of the Invention
The present invention relates to endowing therapeutic protein agents with increased in vivo stability and effectiveness on the central nervous system (CNS). More specifically, the present invention relates to endowing β-glucuronidase protein (GUS) with improved stability in the blood and enhanced ability to affect the CNS, in a therapeutic capacity by attaching a short peptide of acidic amino acids to the N-terminus of the protein.
2. Description of the Related Art
Lysosomal storage diseases (LSDs) are a class of forty rare genetic disorders, each of which is caused by a deficiency in a specific lysosomal enzyme. As a consequence of the progressive accumulation of unmetabolized macromolecules in the lysosomes of cells in various tissues, the disease manifestations worsen over time.1 Individuals afflicted with an LSD can suffer from mild to severe physical and/or neurological abnormalities or can die at an early age. A therapeutic paradigm for the treatment of LSDs was established with the success of enzyme-replacement therapy (ERT) for the treatment of Gaucher disease.2,3 In the case of Gaucher disease, delivery of the enzyme to the affected cells was achieved by modifying the N-linked carbohydrate on the enzyme. This exposed core mannose residues,4,5 enabling the enzyme to bind to the MR, which is highly abundant on cells of the reticuloendothelial system.6,7 These findings led to clinical management of Gaucher disease by ERT.8 Over 3,500 patients have been treated with dramatic clinical results.9 
Meanwhile there is a problem that pharmaceutical preparations of physiologically active proteins like enzymes and peptide hormones are generally made unstable when they are administered to the body, and thus undergo relatively rapid inactivation by, e.g., enzymatic degradation. For pharmaceutical preparations of a physiologically active protein, a method for increasing the stability of the physiologically active protein in the body is known which is based on coupling the proteins to polyethylene glycol.10 
Sly's syndrome is an autosomal recessive, genetic lysosomal storage disease caused by an anomaly in the gene for a lysosomal enzyme, β-glucuronidase (hereinafter referred to as GUS)11(6), and is classified as type VII mucopolysaccharidosis (hereinafter referred to as MPS VII). In lysosomes, GUS acts as an exoglycosidase to remove glucuronic acid residues from the non-reducing termini of GAGs (glycosaminoglycans), such as dermatan sulfate (DS), heparan sulfate (HS), and chondroitin sulfate (CS). In the absence of GUS, GAGs are only partially degraded and accumulate in lysosomes of a variety of tissues. Progressive accumulation of undegraded GAGs in lysosomes affects the spleen, liver, kidney, cornea, brain, heart valves, and the skeletal system, leading to facial dysmorphism, growth retardation, systemic bone dysplasia, deafness, mental retardation, and shortened lifespan.
No effective remedy is currently available for MPS VII. Enzyme replacement therapy (ERT) has been considered to be the potential remedy for MPS VII. Considering its rapid inactivation in the body, however, native GUS is not expected to give any satisfactory effect.
The challenge is to improve joint and brain-related pathology since most of the enzyme-based drugs are delivered to major visceral organs like liver and spleen and only a small amount of enzyme is delivered to bone and brain. Many lysosomal enzymes have a short half-life when injected into the bloodstream because of rapid clearance in the liver by carbohydrate-recognizing receptors, particularly the mannose receptor that is highly abundant on Kupffer cells.12 Although a part of the enzyme reaches the bone marrow, there is no way to guarantee that the enzyme will reach the brain since the blood brain barrier presents a formidable obstacle. As a result, current ERT doses not work efficiently on the bone and brain lesions.
The inventors have sought to address the problem of stability of therapeutic proteins in vivo and the inability of some proteins to effectively cross the blood brain barrier. The inventors have previously disclosed the use of short peptides of acidic amino acids to target proteins to bone tissue for use in Enzyme Replacement Therapy (ERT).13 The inventors have also disclosed the use of short peptides of acidic amino acids to improve stability of physiological active proteins in the blood.
The addition of 4-15 acidic amino acids to GUS results in an increase in molecular weight which generally, would not be expected to increase functional activity of proteins to the CNS. In fact, higher molecular weigh molecules are more effectively excluded from the brain by an ineffectual crossing the blood brain barrier. Similarly, an increase in the hydrophilic nature of a molecule is also thought to exclude molecules at the blood brain barrier. The inventors have made the surprising discovery that despite causing an apparent increase in molecular weight and increase in hydrophilic nature, the addition of an acid amino acid leader to GUS has allowed enhanced therapeutic benefits on the brain.